This patent application claims priority from KO-2001-0057837, the disclosure of which is incorporated by reference herein in its entirety.
The present invention relates to novel peptides with increased + charge and hydrophobicity by substituting one or more amino acids of CA-MA peptide in which cecropin A (CA) and magainin 2 (MA) were conjugated and pharmaceutical compositions containing thereof. More precisely, the present invention relates to synthetic peptides prepared by substituting one or more amino acids of CA-MA peptide represented by the SEQ. ID. NO: 1 with amino acids having + charge and hydrophobicity and anti-bacterial, anti-fungal and anticancer compositions containing thereof. The synthetic peptides of the present invention have no cytotoxicity but have excellent anti-bacterial, anti-fungal and anticancer activity, leading in an effective use thereof as a safe anticancer agent and antibiotics.
Bacteria infection is one of the most common but fatal causes for human diseases. Infection has been successfully treated by antibiotics, but the abuse of antibiotics brought another problem that bacteria now might have resistance against antibiotics. In fact, the speed which bacteria are adapting and having resistance against new antibiotics outruns that of developing new antibiotics analogues. For example, fatal Enterococcus faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa are known to have raised their resistance against every possible antibiotics (Stuart B. Levy, Scientific American, 1998, 46-53).
Tolerance is different from resistance against antibiotics, and it was firstly found in Pneumococcus sp. in 1970s, which provided an important clue for disclosing the mechanism of penicillin (Tomasz, et al., Nature, 1970, 227, 138-140). Some bacteria species having tolerance stopped growing under the ordinary concentration of antibiotics but never died. Tolerance is caused by that the activity of autolytic enzyme of bacteria, like autolysin, is suppressed when the antibiotics inhibit cell wall synthetase. Penicillin can kill bacteria by activating endogenous hydrolytic enzyme; on the other hand, bacteria can survive by restraining the activity thereof even when being treated with antibiotics.
It is a clinical hot issue that bacteria are having tolerance against various antibiotics since infection cannot be effectively cured with antibiotics due to the tolerance (Handwerger and Tomasz, Rev. Infec. Dis., 1985, 7, 368-386). Again, once bacteria have tolerance, they can have resistance, which helps that bacteria survive under antibiotics treatment. Such bacteria can acquire new genetic elements having resistance against antibiotics, thus they can grow even under antibiotics treatment. Actually, bacteria having resistance have tolerance, too (Liu and Tomasz, J. Infect. Dis., 1985, 152, 365-372). Thus, it is urgent to develop novel antibiotics, which can kill antibiotics-resistant bacteria.
There are two types of tolerance in the aspect of its mechanism. The first one is phenotypic tolerance, which occurs when the growing speed decreases in all kinds of bacteria (Tuomanen E., Revs. Infect. Dis., 1986, 3, S279-S291), and the second one is genotypic tolerance acquired by mutation in a certain type of bacteria. For both cases, down regulation of autolysin activation is basically occurring. In the case of phenotypic tolerance acquired by outside stimulus, down regulation takes place temporally while down regulation occurs permanently in the case of genotypic tolerance acquired by mutation, which cause the change of hemolysis regulating routes. Autolysin deficiency is believed to cause the simplest genotypic tolerance, but the bacteria having tolerance acquired by autolysin deficiency have not been reported yet. Such tolerance observed in clinics rather seemed to be caused by the regulation of autolysin activity (Tuomanen et al., J. Infect. Dis., 1988, 158, 36-43).
In order to fight bacteria having tolerance against antibiotics, it is required to develop new antibiotics including one that is working separately from autolysin activity. In addition, it is also required to provide pharmaceutical compositions containing thereof to treat bacteria infection and inflammation effectively.
Meanwhile, bacteria can kill the neighboring bacteria by synthesizing peptides or small organic molecules, which are called bacteriocin. Such bacteriocins are classified into three groups according to their structure. The first group is lantibiotics, the second group is nonlantibiotics, and the third group is those, which are secreted by signal peptide (Cintas et al., J. Bad., 1998, 180, 1988-1994). Animals including insects also produce naturally synthesized peptide antibiotics (Bevines et al., Ann. Rev. Biochem., 1990, 59, 395-414), which are classified into three groups according to their structure as well. The first group is cysteine-rich xcex2-sheet peptides, the second group is xcex1-helical amphiphilic peptides, and the third group is proline-rich peptides(Mayasaki et al., Int. J. Antimicrob. Agents, 1998, 9, 269-280). Those anti-bacterial peptides are known to play an important role in host-defense and congenital immune system (Boman, H. G., Cell, 1991, 65, 205; Boman, H. G., Annu. Rev. Microbiol., 1995, 13, 61). The anti-bacterial peptides have many different structures depending on amino acid sequences, and the most common structure is amphiphilic xcex1-helical structure having no cysteine, just like cecropin, an anti-bacterial peptide found in insects.
Among those peptides, the anti-bacterial activity of amphiphilic peptides has been studied and the development of antibiotics using the amphiphilic peptides has been tried. As of today, magainin 2(MA), cecropin A (CA) and melittin (ME) have been reported as amphiphilic peptides.
Amphiphilic peptides of cecropin group were first found in a fruit fly and later in a silkworm pupa and in pig intestine. While cecropin A was reported to have high anti-bacterial activity but low anti-fungal and anticancer activity (Boman, H. G. and Hultmark, D., Annu. Rev. Microbiol., 1987, 41, 103), magainin 2 was known not to have cytotoxic activity but to have appreciable anti-bacterial, anti-fungal, anticancer and anti-protozoa activity (Zasloff, M., Proc. Natl. Acad. Sci. USA, 1987, 84, 5449). It has been further reported that new synthetic peptides having excellent anti-bacterial, anti-fungal and anticancer activity could be prepared by constructing conjugated peptides by recombination from parts of the sequences of the above two peptides (Chan, H. C., et al., FEBS Lett., 1989, 259, 103; Wade, D., et al., Int. J. Pept. Prot. Res., 1992, 40, 429).
The present inventors have designed and synthesized novel peptides having amino acid sequences with + charge and hydrophobicity at amino terminal, taking amphiphilic peptide conjugated cecropin A and magainin 2 as a template. And the present invention has been accomplished by confirming that the synthetic peptides of the present invention could be effectively used as anticancer agents and antibiotics owing to their anti-bacterial, anti-fungal and anti-cancer activity.
It is an object of this invention to provide novel peptides and their derivatives with increased + charge and hydrophobicity by substituting one or more amino acids of cecropin A and magainin 2 conjugated CA-MA peptide represented by the SEQ. ID. NO: 1 and with excellent anti-bacterial, anti-fungal and anticancer activity without cytotoxicity.
It is another object of this invention to provide pharmaceutical compositions for anti-bacterial, anti-fungal and anticancer agent containing the above synthetic peptides.